Different polypeptide ligands can bind to specific cell surface receptors on the same cell and initiate different intracellular events including the immediate (non-protein synthesis requiring) activation of different sets of genes. We have discovered latent cytoplasmic transcription factors that are activated by such ligands. Those latent cytoplasmic proteins, termed STAT proteins for signal transducers and activators of transcription are phosphorylated on tyrosine in the cytoplasm before translocating to the nucleus to direct transcription. They were first discovered in cells treated with IFN-alpha or IFN-gamma. In this proposal we describe experiments to define the functional domains of one of these proteins, STAT 91, and the functional domains of the kinases (Jak1 and Jak2) that have been shown to be involved in the STAT activation pathway. A most important thrust of future work will be discover other proteins in this same family that serve in response to other ligands. Because the genes encoding the presently known STAT proteins have been found to have many (20) exons we will study the STAT mRNAs in different mouse tissues and in cells treated in a variety of ways to search for differently spliced STAT mRNAs that might function in different ligand-dependent pathways. Several newly discovered STAT protein family members with mRNAs that are present in the thymus are also described that have high homology to but are distinctly different from the already described STAT 91 and 113 proteins. Characterization of these proteins with particular attention to their possible tyrosine phosphorylation in response to other ligands is an important part of this proposal. Finally, collaboration is planned to study the three- dimensional structure of important domains of the STAT proteins and the kinases with which they interact.